Osteoporosis is a systemic disease characterized by low bone mass and microarchitectural deterioration of bone tissue. The current clinical assessment metric, bone mineral density (BMD), sheds little light on microarchitectural deterioration. BMD used in combination with the degree of anisotropy (DA) in the bone microarchitecture is strongly predictive of vertebral strength and stiffness, but critical barriers preclude the clinical assessment of the degree of anisotropy in bone, especially within vertebrae where fractures have a high mortality and are highly predictive of future fractures. A low dose, non-invasive tool capable of measuring DA in vertebrae would overcome these barriers and provide clinicians with a more specific and individualized metric for bone strength and fracture risk. In this application, we propose to develop such a tool based on diffraction enhanced imaging (DEI), a phase contrast x-ray imaging technique. DEI can detect extremely small angular spreading that occurs when an x-ray beam is refracted by microstructures in bone. DEI has directional sensitivity that enables it to measure the angular orientation and the degree of anisotropy in the refracting microarchitecture. In this proof-of-principle study, our goal is to elucidate the relationship between the DEI- measured degree of anisotropy and the strength and stiffness of the vertebrae. In Specific Aim 1, we will establish DEI's ability to measure the degree of anisotropy in vertebrae through comparing the DEI-measured degree of anisotropy to the gold standard measure from micro-CT. Cadaveric vertebrae (age 60-64 male n=3, female n=3; age 70-74 male n=3, female n=3; and ages 80-84 male n=4, female n=4) will be obtained and the BMD will be assessed with a clinical dual-energy x-ray absorptiometry system. Micro-CT images will be used to measure bone morphology parameters of each vertebra. A series of DEI reflectivity profile images will be obtained and these images will be combined into 2D images mapping the preferred orientation direction and DA at each position in the vertebra. Linear regression analysis will be used to elucidate the mathematical relationship between gold standard bone measurements and the DEI-based measures. In Specific Aim 2, we will elucidate the relationship between vertebral strength and stiffness and the DEI-measured degree of anisotropy. Biomechanical testing will be performed to assess the strength and stiffness of the vertebrae. The correlation coefficient between the stiffness and strength of each vertebra and the DEI-measured DA will be calculated. Multiple regression analysis will determine the effectiveness of the DEI-measures in combination with BMD for assessing bone strength. In this study, we will establish DEI's sensitivity to DA in vertebral microarchitecture and validate a nove microarchitectural assessment tool for predicting fracture strength and stiffness in intact vertebrae. DEI's unique capability for assessing microarchitectural deterioration at otherwise non-resolvable size-scales will give clinicians a powerful new tool for assessing fracture risk. These findings will drive multiple future studies where we will translate this technology into the clinic.